【摘要】：目的 骨髓間充質(zhì)干細(xì)胞（bone marrow mesenchymal stem ceIIs,BMSCs）移植治療大腦中動脈阻塞(Middle CerebralArtery Occlusion，MCAO)模型鼠后，在不同時間點采用western blot、 ELISA方法檢測IL-1β、TNF-α的表達(dá)變化，分析BMSCs移植對缺血性腦卒中后炎性因子的影響，探討B(tài)MSCs移植治療缺血性腦卒中修復(fù)受損神經(jīng)功能的可能抗炎機(jī)制。 方法 取幼年雄性SD大鼠骨髓腔細(xì)胞，通過全骨髓培養(yǎng)法和細(xì)胞貼壁培養(yǎng)法體外分離、培養(yǎng)、擴(kuò)增得到BMSCs，傳至第4代，免疫熒光法鑒定，誘導(dǎo)分化為成骨細(xì)胞、脂肪細(xì)胞及神經(jīng)干細(xì)胞并鑒定。取成年雄性SD大鼠，用Longa線栓法制造MCAO模型。參考Bederson JB評分法測評大鼠神經(jīng)功能缺損，篩選入組MCAO模型鼠；TTC染色法顯示腦組織梗死范圍，剔除TTC（-）或有出血的大鼠樣本。 取造模成功的MCAO模型鼠完全隨機(jī)分為2組：A組/MCAO模型組（n=16），即建立MCAO模型后注射1ml生理鹽水；B組/實驗組（n=16），即建立MCAO模型后通過尾靜脈注射法移植1ml2×l09/L BMSCs；A、B組各分為處理后1d、3d、7d、14d時間點亞組（n=4）。取正常大鼠設(shè)C組/空白組（n=4），不予任何干預(yù)。各亞組行心臟取血，ELISA方法檢測大鼠血清IL-1β、TNF-α的表達(dá)變化；生理鹽水灌注取腦，western blot方法檢測大鼠腦組織IL-1β、TNF-α的表達(dá)變化。 結(jié)果 1.成功培養(yǎng)得到BMSCs。鏡下細(xì)胞形態(tài)：原代BMSCs呈圓形或類圓形，貼壁數(shù)量少；經(jīng)過純化、擴(kuò)增，貼壁細(xì)胞增多，呈長梭形，集落樣生長，局部呈漩渦狀；至P7代，細(xì)胞逐漸老化，以扁平細(xì)胞為主。細(xì)胞免疫熒光法鑒定：CD54（+）、CD34（-），符合BMSC免疫表型特點。BMSCs誘導(dǎo)為成骨細(xì)胞見細(xì)小顆粒于培養(yǎng)液；誘導(dǎo)為脂肪細(xì)胞見紅色脂滴于胞漿；誘導(dǎo)為神經(jīng)干細(xì)胞見棕色顆粒于胞漿。 2.大鼠神經(jīng)功能評分：造模后1天實驗組評分較模型組無明顯差異（P0.05），3天、7天、14天實驗組較模型組均有下降（P0.05），差異有統(tǒng)計學(xué)意義。 3.大鼠血清IL-1β、TNF-α表達(dá)（單位：ng/L）：1天、3天、7天、14天各亞組：IL-1β濃度為模型組27.0±0.35、29.2±0.60、25.2±0.65、22.8±0.66，，實驗組25.2±0.31、26.8±0.56、20.8±0.39、19.2±0.94，空白組10.9±0.54；TNF-α濃度為模型組231±1.46、225±4.98、216±4.16、198±4.61，實驗組189±2.85、178±3.17、166±5.27、157±6.03，空白組146±6.86；模型組較空白組均明顯增多（P0.05），實驗組較模型組均減少（P0.05），但較空白組仍增多（P0.05）。模型組及實驗組與空白組比較：IL-1β表達(dá)1天增多，3天達(dá)高峰，7天下降，14天持續(xù)下降，但仍高于空白組（P0.05）；TNF-α表達(dá)1天增多達(dá)高峰，3天下降，7天、14天緩慢下降，但仍高于空白組（P0.05），差異有統(tǒng)計學(xué)意義。 4.大鼠腦組織IL-1β、TNF-α表達(dá)（灰度值）：1天、3天、7天、14天各亞組：IL-1β為模型組5502±108.9、6267±160.5、4425±158.1、3352±187.1，實驗組4217±143.7、4706±111.8、3235±150.7、2307±120.7，空白組1874±70.7； TNF-α為模型組7801±148.9、6296±170.7、5399±240.9、3458±180.2；實驗組6468±180.9、5789±119.7、4647±207.3、2828±152.6，空白組2215±159.5；模型組較空白組均增高（P0.05），實驗組較模型組均降低（P0.05）較空白組增高（P0.05）。模型組及實驗組與空白組比較：IL-1β表達(dá)1天增高，3天達(dá)高峰，7天下降，14天持續(xù)下降但仍高于空白組（P0.05）；TNF-α表達(dá)1天增高達(dá)高峰，3天下降，7天、14天緩慢下降，但仍高于空白組（P0.05），差異有統(tǒng)計學(xué)意義。 結(jié)論 1.缺血性腦卒中模型鼠中IL-1β、TNF-α表達(dá)增加，其神經(jīng)損傷可能與炎癥反應(yīng)有關(guān)。 2.骨髓間充質(zhì)干細(xì)胞移植可降低MCAO模型鼠IL-1β、TNF-α的表達(dá)，其修復(fù)神經(jīng)功能的作用可能與BMSC下調(diào)炎性因子減輕炎癥反應(yīng)有關(guān)。
[Abstract]:objective
After transplantation of bone marrow mesenchymal stem cells (BMSCs) for treatment of middle cerebral artery occlusion (MCAO) model mice, the expression of IL-1 beta and TNF-a was detected by Western blot and ELISA at different time points. The effect of BMSCs transplantation on inflammatory factors after ischemic stroke was analyzed. Objective to investigate the possible anti inflammatory mechanism of BMSCs transplantation in repairing damaged neural function after ischemic stroke.
Method
Bone marrow cavity cells of young male SD rats were isolated, cultured and amplified in vitro by whole bone marrow culture and cell adherence culture. BMSCs were identified by immunofluorescence. The adult male SD rats were induced to differentiate into osteoblasts, adipocytes and neural stem cells and identified. MCAO model was established by Longa thread plug method. Neurological deficits were assessed by on-JB scoring method, and MCAO model rats were selected. The infarct size of cerebral tissue was shown by TTC staining, and TTC (-) or bleeding rat samples were excluded.
MCAO model rats were randomly divided into two groups: group A / MCAO model group (n = 16), 1 ml normal saline was injected after establishing MCAO model; group B / experimental group (n = 16), 1 ml 2 xl09 / L BMSCs were transplanted by tail vein injection after establishing MCAO model; group A and group B were divided into 1, 3, 7 and 14 day subgroups after treatment (n = 4). No intervention was given to the control group (n=4). Blood samples were taken from the heart of each subgroup. The expression of IL-1beta and TNF-alpha in serum was detected by ELISA, and the brain was perfused with normal saline, and the expression of IL-1beta and TNF-alpha in brain tissue was detected by Western blot.
Result
1. BMSCs were successfully cultured. The morphology of primary BMSCs was round or quasi-circular, with few adherent cells; after purification, amplification, adherent cells increased, spindle-shaped, colony-like growth, local vortex; to P7 generation, cells gradually aging, mainly flat cells. Identification of cell immunofluorescence: CD54 (+), CD34 (-), in line with BMSC immunity. BMSCs were induced into osteoblasts with fine granules in culture medium, adipocytes with red lipid droplets in cytoplasm, neural stem cells with brown granules in cytoplasm.
2. Rat neurological function score: There was no significant difference between the experimental group and the model group on the first day after modeling (P 0.05). The scores of the experimental group on the third day, the seventh day and the fourteenth day were lower than those of the model group (P 0.05).
3.The expression of IL-1betand TNF-alphain serum of rats (unit: ng/L):1 day, 3 days, 7 days, 7 days, 14 days:1 day, 3 days, 7 days, 14 days:the concentration of IL-1 Betin the model group was 27.0 [0.35, 29.2 [0.60, 25.2 [0.60, 25.2 [0.65, 22.2 [0.65, 22.8 [0.66, 22.8] 0.66, experimental group 25.2 [0.2 [0.31, 26.8 [0.56, 20.8 [0.39, 19.39, 19.2 [.2 [0.94.94] 94, blankgroup 10.9 198 + 4.61, experimental group Compared with the blank group, the expression of IL-1 beta in the model group and the experimental group increased in one day, reached a peak in three days, decreased in seven days and continued to decline in 14 days, but still higher than the blank group (P 0.05). In the blank group (P 0.05), the expression of TNF-alpha reached the peak in one day, decreased in three days, slowly decreased in seven days and 14 days, but still higher than the blank group (P 0.05), the difference was statistically significant.
4.Expression of IL-1 beta and TNF-alphain rat brain tissues (gray value):1 day, 3 days, 7 days, 7 days and 14 days:1 day, 7 days, 14 days:1 day, 3 days, 7 days, each subgroup:IL-1 betwas 5502 [108.9, 6267 [160.5, 4425 [160.5, 4425 [158.5 [158.1, 3352 [188.1, 3352 [187.1,3352].1.1,3352 [[4 17 [143.7.7.7,4706 [111.8, 4706 [111.8, 3235 [150.7, 2307 [150.7, 2307 [180.2; solid Compared with the blank group, the expression of IL-1 beta in the model group and the experimental group increased in one day, reached a peak in three days, decreased in seven days, but remained higher in 14 days. In blank group (P 0.05), the expression of TNF-alpha increased as high as 1 day, decreased on 3 days, decreased slowly on 7 days and 14 days, but still higher than blank group (P 0.05), the difference was statistically significant.
conclusion
1. The expression of IL-1beta and TNF-alpha increased in ischemic stroke model rats, and the nerve injury may be related to inflammation.
2. Bone marrow mesenchymal stem cell transplantation can reduce the expression of IL-1beta and TNF-alpha in MCAO model mice, and its effect on repairing nerve function may be related to the reduction of inflammatory factors by BMSC.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3

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